Discovering novel therapeutic small molecules using conventional in vitro approaches requires a prior selection of an appropriate molecular target. However, in the case of cyanide, it is not obvious which biological pathways must be targeted by small molecules to effectively reverse the toxicities of chemical exposure. In addition, in vitro approaches to small molecule discovery fail to predict a compound's bioavailability, ADME properties (adsorption, distribution, metabolism, excretion), and toxicity. One promising alternative approach for discovering novel cyanide countermeasures is the use of whole-organism, phenotype-based chemical screens. If the physiological effects of cyanide could be monitored in an assay that is also amenable to high-throughput screening (HIS)/ novel small molecules could be identified that reverse the effects of cyanide. We have developed a zebrafish model in which exposure to cyanide causes stereotypical toxicities including bradycardia, neuronal necrosis, and death. By subjecting this model to high-throughput screening, we propose to identify small molecules that prevent or reverse cyanide toxicity through a variety of novel mechanisms. Cyanide-exposed zebrafish will be treated in 96-well plates with compounds from large, structurally diverse chemical libraries. The libraries will contain uncharacterized compounds as well as those with known biological activities (including FDA-approved drugs). By varying the screening conditions, we propose to discover a range of agents that accelerate post-exposure recovery, in addition to novel detoxifying agents. Once promising lead compounds are discovered, we will use structure activity relationship (SAR) studies to improve compound potency and to minimize unwanted side effects. The goal of this proposal is the generation of at least three novel optimized leads of cyanide countermeasure suitable for advanced preclinical efficacy and safety testing. Specifically, we propose: Aim 1. To determine the efficacy of known countermeasures in a zebrafish model of cyanide exposure. Aim 2. To identify novel chemical classes of cyanide countermeasure through high-throughput in vivo screening. Aim 3. To optimize the most promising classes of lead compound for potency and safety.